Cell therapy with bone marrow stem cells (BMSCs) remains a viable option for tissue repair and regeneration. One major challenge for cell-based therapy is the poor survival of the cells after transplantation. The exact mechanism(s) for impaired survival of the implanted BMSCs remains to be defined. MG53 is an important cell membrane repair protein that is naturally produced in skeletal and cardiac muscles, and present in blood that mediates the tissue protective function for MG53. Oxidized low-density lipoprotein (ox-LDL) is a natural product in blood. We observed that ox-LDL at the concentrations compatible with the serum ox-LDL levels in patients with coronary artery disease produced significant membrane damage to BMSCs as reflected by the release of lactate dehydrogenase (LDH) from the cell and the entry of fluorescent dye FM1-43 into the cell. Treatment with recombinant human MG53 (rhMG53) protein could reduce LDH release and FM1-43 dye entry in BMSCs exposed to ox-LDL. Further in vivo studies showed that intravenous ox-LDL administration led to decreased serum MG53 level, and impaired survival of BMSCs implemented into normal C57B6 mice. The proposed study will test the hypothesis that ox-LDL impairs the survival of BMSCs through direct damage to their cell membrane, and ox-LDL-mediated reduction of MG53 level in circulation is an important factor for the limited survival of BMSCs with compromised cell membrane repair capacity and reduced efficacy of cell-based therapy. The specific aims are: 1) to determine the effect of ox-LDL on the membrane integrity of BMSCs both in vitro and in vivo; and 2) to define the mechanism(s) for ox-LDL-mediated action on MG53 and the survival of BMSCs in cell transplantation. We will elucidate the mechanisms that underlie the ox-LDL-induced damage to BMSCs in culture system, and also conduct experiments to evaluate the protective effect of rhMG53 on BMSCs following mechanical or hypoxia-induced membrane injuries. We will further engineer BMSCs that express secretary MG53 to determine if sustained MG53 production has beneficial effects on the survival of BMSCs in vivo in ox-LDL-treated mice and in hyperlipidemic animals. Efforts will be made to determine the mechanism on how ox-LDL causes impairment in MG53-mediated membrane repair. We will determine whether ox-LDL causes decreased MG53 secretion from muscle cells or increased its clearance from circulation. We will further test the hypothesis with the use of MG53 knockout mice and transgenic mouse models where inducible and reversible MG53 expression in circulation can be tailored with a doxycycline-driven promoter system. These studies will reveal the role for MG53-mediated membrane repair in cell-based therapy, provide important information on the molecular mechanisms for the poor survival of implanted BMSCs, and help to explore new strategies like targeting ox- LDL and circulating MG53 to optimizing the survival of BMSCs and enhancing the efficacy of cell-based therapy for cardiovascular diseases. 1